The three aromatic amino acid hydroxylases are responsible for the metabolism of excess ditary phenylalanine, and initiating the biosynthesis of epinephrine and the catecholamine and serotonin neurotransmitters. The reactions are unique in that they are served by a coenzyme, tetrahydrobiopterin, that has no other known function. Using primarily the more abundant phenylalanine hydroxylase as the model enzyme, the objective of this study is to determine in which ways the structure of the reduced pterin is specifically suited to the hydroxylation of three rather inert substrates, and in particular the manner in which it directs the activation of molecular oxygen. It has been shown that the initial step in this process involves covalent addition of O-2 to position C4a of cofactor. The objectives are approached with five specific aims. (1) Cofactor analogs have been designed to determine the structure of the complex which is ultimately responsible for oxidation of substrate. (2) The autooxidations of cofactor will be studied with isotope labeling techniques to determine the extent to which this provides a model for the hydroxylase. (3)\Enzymatic reaction conditions will be modified to allow accumulation of otherwise transient intermediates which can then be observed spectrally, and characterized. (4) We have proposed that the properties of the 5-nitrogen of tetrahydrobiopterin are significantly different from those of N5 of dihydroflavin. A pyrimidine cofactor analog will be used to study the function of this atom in catalysis. (5) The aromatic amino acid hydroxylases contain iron and the role of this metal will be studied. An understanding of the mechanism of these enzymes will allow the design of cofactor analogs capable of regulating their activity in vivo.